Homogeneous fluoroimmunoassay involving autocorrelation processing of optically sensed signals

ABSTRACT

In the course of a reaction in which one of the reactants is on the surface of carrier particles in a solution and another of the reactants is tagged with a fluorescent substance, some of the fluorescently tagged reactant attaches to, or is displaced from the carrier particle. The present invention relates to a method and device for determining the amount of fluorescently-tagged reactant which is attached to the carrier particle or which is free in solution, without physically separating the carrier particles from the solution. In a particular application of the invention (immunoassay) the reaction is between antibodies and antigens, and from the amount of fluorescently-tagged reactant which is attached to the carrier particle one can determine the unknown amount of antigen in a sample. The number of fluorescent particles which are bound to the large carrier particles and which are in solution are determined by optical and electronic sensing and analyzing techniques, including (1) shadowing techniques in which large opaque carrier particles block the pick-up of fluorescent radiation from bound tagged reactants, and/or (2) autocorrelation techniques which selectively discriminate between radiation from the bound fluorescently tagged reactants on the large particles and the free fluorescently-tagged reactants which have a more rapid diffusion movement than the carrier particles.

This patent application is a continuation-in-part of patent applicationSer. No. 194,856, filed Oct. 7, 1980.

FIELD OF THE INVENTION

The present invention relates to process analysis or immunoassaytechniques.

BACKGROUND OF THE INVENTION

In connection with the diagnosis and treatment of certain healthproblems, it is most useful to identify and measure the variousantibodies, drugs, proteins and other "macromolecules" in various bodyfluids. One of the most widely used techniques for making suchdeterminations is the radioimmunoassay. The history of radioimmunoassaydevelopment is set forth, for example, in an article entitled "APhysicist In Biomedical Investigation", by Rosalind S. Yalow, PhysicsToday, October 1979, pages 25-29. The principal disadvantages ofradioimmunoassays are their reliance on expensive and potentiallyhazardous reagents which possess a limited shelf life, the specialhandling and disposing procedures which are required for radioactivematerial, and the expensive instrumentation which is needed.

There are also several fluorescence-based immunoassay techniques whichare currently in use or are undergoing clinical evaluation, with one ofthese techniques being described in a booklet entitled "Immuno-fluor",and subtitled "Quantitative Immunofluorescent Assay for Human GammaGlobulin", May 1978, and originating with Bio-Rad Laboratories. In onetype of radiation and fluorescent immunoassay technique which has beenproposed, relatively large carrier particles are coated with one of theactive materials, usually the antibody, a sample to be tested is addedto the solution, and the antigen in the sample is bound to the antibody.A fluorescently labeled antibody is then added to the mixture and bindsto the antigen. The amount of fluorescently labeled antibody which isattached to the antigen, and is therefore bonded to the larger particlesis directly proportional to the amount of the antigen in the sample. Theexcess fluorescently labeled antibody is then separated by standardcentrifuging and decanting techniques. The remaining material consistssubstantially of the large particles and the attached (1) antibody, (2)antigen, and (3) fluorescently tagged antibody. The level offluorescence of this residual material indicates the level of antigenpresent in the sample under test. This reaction is called a "sandwich"reaction because the antigen is sandwiched between the antibody coatedto the carrier particle and the fluorescently labeled antibody.

In another immunoassay technique, carrier particles are also coated withan antibody. The coated particles, the sample with an unknown amount ofantigen, and a known amount of tagged (either radioactive orfluorescent) antigen are placed in solution. The unknown antigen and thetagged antigen then compete for binding to the antibody. The carrierparticles are then separated out of the solution and the amount oftagged antigen on the carrier particles is a measure of the amount ofunknown antigen in the sample, the more tagged antigen bound to thecarrier particles the less the unknown antigen in the sample. This typeof reaction is called a competitive reaction, since the tagged antigenand unknown antigen compete for binding to the antibody which isattached to the carrier particles.

The foregoing procedures are employed in a number of radioactive-basedand fluorescent-labeled assays. In each case, however, the analysis ismade in the course of a reaction in which radioactive orfluorescent-tagged substances are either bound to larger particles orare shifted into solution from previous sites on the larger particles.Subsequently, the larger carrier particles are centrifuged, the residualliquid is decanted, and this is sometimes followed by additionalpurification steps in which liquid is added and another centifuging stepis undertaken to insure the removal of any unbound component which mightotherwise affect the assay signal obtained from the residue associatedwith the larger carrier particles.

While the prior assay techniques have proved very accurate, and arewidely used, a large amount of time is spent in the separation steps;and in the case of the radioassay methods, the problem of handling theradioactive material is troublesome, and of course the undesired extraexposure to radiation is unfortunate, with its adverse healthimplications.

Accordingly, a principal object of the present invention is to providean assay or analysis method which does not require the use ofradioactive material, and also which does not require the physicalseparation of the carrier particles from the solution.

SUMMARY OF THE INVENTION

In accordance with one specific aspect of the invention, a fluorescentimmunoassay technique includes the use of relatively large carrierparticles with at least one active component coated on these particles,and the exposure of the particles to at least two additional activecomponents in solution. The carrier particles may be inert particles orthey may be human or animal blood cells, or other biological cells withan active component (which may be natural) on their surface. With one ofthe active components being fluorescently tagged, there is a shifting ofsome of the tagged components from solution where they are free, into astate where they are bound to the carrier particles. Subsequently, thesolution (without any separation) is subjected to intense illuminationwhich causes the fluorescently tagged materials to emit light, andoptical and electronic techniques are employed to accurately determinethe number of the fluorescently tagged components which have becomebound to the carrier particles, or which are "free" in solution.

In accordance with one method employed to determine whether thefluorescent particles are "bound" to the larger particles or are "free",the faster diffusion rate of the "free" components as compared withthose which are bound to the slower moving carrier particles, isemployed to discriminate between fluorescent light originating with thetwo different types of particles. In one specific system, anautocorrelator is employed to suppress the signals from the fastermoving "free" particles. Another technique which can contribute to thediscrimination is to use carrier particles which are relatively opaqueto both the incident illumination and the emitted fluorescent light. Inthe forward direction (direction of the incident illumination), thefluorescent light from molecules bound to the carrier particles will be"shadowed" by the opaque carrier particles compared with the lightemitted in the backward direction, whereas fluorescent light from freemolecules will not be shadowed in the forward direction. The differencebetween the fluorescent light measured at different angles with respectto the incident illumination can be used to disciminate betweenfluorescence arising from molecules bound to the opaque carrierparticles and free molecules.

Another aspect of the invention involves the relative movement of thesample and the optical sensing path to sense a large number of differentvolumes within the solution, thereby improving the accuracy of theassay. Also, the technique of scanning a number of different equalvolume samples in a solution, with each volume only being scanned onceis essentially equivalent to sitting at a fixed location and takingsuccessive samples at successive intervals of time; but the scanningapproach has the advantage of more rapid completion of the analysis.Increased accuracy may be obtained by repetitive successive scanning andanalysis of a large number of different samples. Filters are also usefulin discriminating against the input radiation of one optical wavelengthand restricting the pickup to the fluorescent output light of adifferent color or wavelength.

Other objects, features and advantages will become apparent from aconsideration of the following detailed description and from theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1, 2a and 2b are diagrammatic showings of one fluorescentimmunoassay process;

FIG. 3 is a block diagram of a typical system for the selectivedetermination of bound and free fluorescent material in a sample;

FIG. 4 is a diagrammatic showing of one apparatus which has beensuccessfully tested;

FIG. 5 is a diagrammatic showing of an alternative arrangement in whichthe light source and sensor probe is physically moved relative to thesample;

FIGS. 6a, 6b, and 6c are diagrams schematically depicting another typeof immunoassay reaction;

FIG. 7 shows the capability of the auto-correlation system todiscriminate against the presence of a high level of radiation from freeparticles as compared with the radiation from the bound fluorescentmaterial on the larger carrier particles;

FIG. 8 is a diagrammatic showing of a shadow technique for determiningthe proportion of free and bound fluorescent material in the sample; and

FIGS. 9 and 10 indicate schematically correlation function values as afunction of the sampling intervals, for different sets of conditions.

DETAILED DESCRIPTION

The principles of the present invention will now be considered relativeto an immunoassay, as illustrated in FIG. 1. In this process, one startswith relatively large carrier particles as shown at 22 in FIG. 1 whichmay be made of polystyrene, latex, sephedex beads, glass spheres or somesimilar material. These carrier particles may also be biological cells,such as blood cells. These particles 22 as represented in FIG. 1 arecoated with a given antibody, represented in FIG. 1 by the letter "A",usually by adsorption or covalent bonding. In the case of cells, theantibodies on the surface many be coated on, or may be naturallypresent.

The next step as shown in FIG. 2a involves the addition of an unknownantigen sample which is to be tested along with a known amount of afluorescently tagged antigen which is specific to the antibody A. Wedesignate the unknown antigen by the symbol "U" and the tagged antigenby the symbol "T". Assuming that the unknown antigen is also specific tothe antibody, the "U" and "T" antigens will compete for binding to theantibody as shown in FIG. 2b. The amount of T which binds to the carrierparticles will depend on the amount of U in solution, since they arecompeting for the same binding sites. The more unknown antigen U thereis in solution, the less of the known tagged antigen T that will bind tothe carrier particles. The amount of T which binds to the carrierparticles, or conversely, which stays free in solution, is a measure ofthe amount of unknown antigen U in the solution.

The solution is illuminated with a light beam whose band of wavelengthsis below the band of wavelengths emitted by the fluorescent tag. Forexample, the fluorescent dye fluorescein emits in the yellow-greenportion of the visible spectrum when excited by radiation in the blueregion of the visible spectrum. This light beam could be produced by alaser or by filtering the light from a broad-band source, such as atungsten light, through an optical filter.

Fluorescent light which is emitted from the fluorescent tags is detectedby one or more optical sensors, usually photomultiplier tubes, andfilters can be placed in front of these sensors so that they aresensitive only to the fluorescent light and not the incident excitinglight which is at shorter wavelengths.

Various methods have been developed for determining the amounts of freeand bound fluorescently-tagged molecules which are present, withouttheir physical separation. These methods fall into two classes: (1)discrimination between the fluorescence from tagged molecules bound tothe carrier particles and the fluorescence of either tagged molecules orimpurities which are "free" in solution due to the slower rate ofdiffusion and greater brightness of the carrier particles relative toindividual antibody and/or antigen molecules in solution, and (2)altering the angular distribution of fluorescent light emitted by thebound molecules by using opaque or semi-opaque carrier particles. Thesemethods will be discussed in some detail below.

FIG. 3 is a block diagram of the apparatus in accordance with thepresent invention which is employed to determine the progress of areaction such as that shown in FIGS. 2a and 2b. More specifically, theprocess sample 32 to be tested is irradiated with illumination from thelight source 34 with a band of wavelengths near the peak absorbancewavelength of the fluorescent material. Light from the excitedfluorescent particles or molecules will be picked up by photodetectors36 and 38 along paths indicated at 40 and 42, respectively. Filters 44and 45, which transmit the longer wavelength light from the excitedfluorescent particles, but which block the shorter wavelengthillumination from light source 34, are provided at the inputs to thephotodetectors 36 and 38.

Signals from one or both of the photomultipliers 36 and 38 aretransmitted to the data processing unit 46. In one particular embodimentof the invention only one photodetector is used. The data processingunit 46 may include suitable data processing circuitry for performingcertain calculation functions as will be described below, includingautocorrelation data processing, and will provide control signals overlead 50 to control the relative position of the optical channel and thesample 32, by arrangements indicated by block 52 in FIG. 3. This mayeither involve shifting the light beam relative to the sample, orphysically moving the sample relative to the optical system as disclosedhereinbelow. The output from the data processing unit 46 is indicatedschematically by arrow 54. This may be a digital or varying amplitudesignal, or a printout, as desired. The controller 46 may be providedwith two inputs, or only one, and may be operated either in a mode usingthe autocorrelator capability (normally with only one input) or in theshadow detection mode (normally without autocorrelation), as more fullydiscussed below.

FIG. 4 is a diagrammatic showing of one implementing apparatus which wasactually employed. In FIG. 4, blue light was transmitted from an argonlaser (wavelength equal to 488 mm) along a path 58 through pinhole 60 toimpinge on the sample 62. The yellow-green illumination radiated by thefluorescing particles was picked up by the optical system including lens64, filter 66, and the slit 68 to impinge on the photomultiplier tube70. The output from photomultiplier 70 is processed by a data processorsuch as unit 46 of FIG. 3. The position of the sample 62 was shifted bythe use of a loud speaker cone actuating arrangement 74 to which thesample 62 was attached. Input signals supplied over lead 76 from thedata processing and control circuitry successively and repetitivelyshifted the position of the sample 62 so that different volumes weresampled. As indicated to the right in FIG. 4, the cylindrical samplevolumes may be in the order of 100 microns in length and 100 microns indiameter.

FIG. 5 indicates schematically one form of arrangement employing fiberoptics, where the light source and pickup element are shifted relativeto the sample, rather than vice versa. In FIG. 5 the thin sample cell 82is shown mounted in a liquid container 84 to match the refractive indexof the movable fiber optic element 86, thereby reducing reflections andincreasing optical efficiency. The optical fiber or bundle of fibers 86is optically coupled to two other optical fibers or bundles of fibers 88and 90. Incident light, for example blue light, enters into the opticalfiber conduit 88 and is conducted to the sample 82 by the optical fiberconduit 86. Fluorescing molecules in sample 82 provide output radiationwhich is picked up and transmitted over the optical fiber conduit 90.The filter 92 blocks the blue light from the original source buttransmits the fluorescent radiation, which might for example be yellow,through to the detector 94 which can be any suitable photo detector.

Turning now to FIG. 6, the three FIGS. 6a, 6b and 6c show successivesteps in another immunoassay process. More specifically, FIG. 6a shows alarge particle 102 of the type shown in FIGS. 1 and 2, with the letters"A" indicating antibodies on the beads 102 which may be plastic beads orcells, as mentioned above. The result as shown in FIG. 6a is a stableimmunoadsorbent.

A sample containing an unknown amount or concentration of antigen,identified by the letter B, is added to the solution, and the resultingsolution incubated at optimum temperature for a period of time. All ofthe antigen B is bound to the solid phase immunoadsorbent, because theimmunoadsorbent is maintained in excess. This situation is showndiagrammatically in FIG. 6b.

FIG. 6c shows the situation following the addition of a fluorescentlylabeled antibody, which is specific to the antigen B, designated by theletter "F". Each site, as designated with the letter B symbol, hasassociated with it a tagged monospecific antiserum designated "F",because this antibody is added in excess. Accordingly, the fluorescentmaterial which binds to the large particles is directly proportional tothe amount of antigen supplied in the step shown in FIG. 6b; and thereis also some additional free fluorescently tagged antibody F, asindicated by the letters "F" which are spaced apart from the B symbolsin FIG. 6c.

Up to the point shown in FIG. 6c, the process is substantially thatcurrently carried out commercially. In the commercial processes, thenext step is to separate the unbound fluorescently tagged antibody "F"by centrifuging, and discarding the supernatant and resuspending thespun-down material in a non-fluorescing solvent, sometimes in severalsteps to insure purity, and then measuring the fluorescence of theresidue. However, as mentioned hereinabove, these successive steps inthe physical separation are time consuming and, in the aggregate, quiteexpensive in view of the manpower and skilled technician time which isrequired.

In the balance of the present specification, the techniques which havebeen developed by the inventors for distinguishing between bound andfree fluorescently tagged material, and therefore determining theconcentrations of the unknown solutions being tested, without physicalseparation, will be set forth. While the techniques to be described areapplicable to a variety of systems, our discussion will relate primarilyto the immunological applications of the type discussed broadlyhereinabove. We will now proceed with a simplified mathematical analysisof the principles underlying the proposed techniques.

Let us assume that the solution contains N identical fluorescingparticles per sampled volume δV. At any instant the number of particlesin δV will fluctuate according to Poisson statistics, where the rootmean square (r.m.s.) magnitude of the fluctuations is equal to thesquare root of N. A convenient way to monitor these fluctuations is toevaluate the familiar intensity autocorrelation function,

ti C(t)=<I(t')·I(t'-t)>_(t')

where I(t') is the fluorescent intensity originating from δV at time t'and the symbol < . . . >_(t') indicates an average of the intensityproduct over a large number of samples.

The usefulness of the autocorrelation function in our applicationdepends upon the fact that a number fluctuation in δV has a finitelifetime, where τ is the mean time for diffusion of the fluorescingparticles out of (or into) the volume δV. This diffusion time isdirectly proportional to the hydrodynamic radius of the particle; thelarger the particle, the longer the diffusion time and therefore thelonger the persistence time of fluctuations in particle number in volumeδV.

The shape of the intensity autocorrelation function as a function of thetime between samples, t, is shown schematically in FIG. 9. In general,the correlation function is large for small values of t, and thendecreases to some baseline when t becomes larger than τ; at times t>>τ,there is no correlation between fluctuations in intensity for successivemeasurements since the number fluctuations will have "diffused away"between measurements. Therefore the correlation function would give justthe square of the average intensity. If we assume that each particlefluoresces with intensity I, then the average intensity is N.I and thebaseline of the correlation function is (N·I)². At very short sampletimes, t<<τ, the number fluctuations persist between intensitymeasurements. If we assume that the mean fluctuation in the number ofparticles about the average N is √N, then the autocorrelation functionat short sample times, averaged over many samples, will be I² N² +I² N;i.e. I² N above the baseline.

This would also be true for the special case of zero sampling time (t=0)where the correlation function is

    C(0)=<I(t')I(t')>.sub.t' =<I(t').sup.2 >.sub.t'

which is just the square of the intensity measured at time t' averagedover a large number of samples.

If the solution contains two species of fluorescent particles which areof different size, the correlation function would look somewhat likethat shown in FIG. 10. The smaller particles have a diffusion time τ_(s)and the large particles have a diffusion time τ_(l). We assume that theaverage number of large particles and small particles in the samplevolume is N_(l) and N_(s), respectively, and that their respectivefluorescent intensities per particle are I_(l) and I_(s). Assumingstatistical independence of the two types of particles, one obtains thefollowing values for the correlation function in three separate regionsshown in FIG. 10.

    ______________________________________                                        region 200 t >> τ.sub.l                                                                     C(t) = B = baseline                                         region 202 τ.sub.l > t > τ.sub.s                                                        C(t) = B + N.sub.l I.sub.l.sup.2                            region 204 t << τ.sub.s                                                                     C(t) = B + N.sub.l I.sub.l.sup.2 + N.sub.s I.sub.s.sup.2    ______________________________________                                    

One may then measure the correlation function at different samplingtimes and separate the fluorescence from the two different sizedparticles. In the immunoassay, the large particles would be the carrierparticles with the fluorescence from them being due to the fluorescencefrom the tagged molecules bound to the carrier particles, and the smallparticles would be the tagged molecules not bound to the carrierparticles. By measuring the autocorrelation function at differentsampling times, one can separate the fluorescence from the boundmolecules and the fluorescence from the free particles. If one wereinterested in measuring the fluorescence from the bound molecules, onewould measure the correlation function in the region 202 and in thebaseline region 200, and subtract the two results to obtain a measure ofthe amount of tagged reactant bound to the carriers. To obtain a measureof the tagged reactant which is not bound to the carrier particles (i.e.that which is "free" in solution) one would measure the correlationfunction in the region 204 and in the region 202, and subtract theresults.

The correlation function has the property that its magnitude abovebaseline depends on the square of the intensity from each particle.Therefore bright particles contribute much more to the correlationfunction than dim particles. Let us say, for example, that the number offree fluorescent molecules is equal to the number of fluorescentmolecules bound to the carrier particles, but that there are on theaverage 10 fluorescent molecules bound to each carrier particle. Underthese assumptions then, there are 10 times as many free fluorescentmolecules than carrier particles in a sampling volume. Although thetotal fluorescent light from the bound and free fluorescent molecules isthe same, the bound molecules are weighted more heavily in thecorrelation function. For our example, N_(s) =10 N_(l) I_(l) =10 I_(s)and in region 204 of FIG. 14, the contribution to the correlationfunction from the bound fluorescence is 10 times the contribution fromthe "free" fluorescence. So, independent of the diffusion rates of thebound and free fluorescence, the correlation function emphasizes thefluorescence from the bound molecules when, on the average, more thanone fluorescent molecule binds to each carrier particle.

For instance, the special case of the correlation function at zerosampling time, <I(t')² >_(t'), could be used to detect changes in theratio of the free to bound fluorescence. As the fluorescence goes, say,from the free state to being bound on the carrier particles, thisfunction, minus the baseline, would increase as the carrier particlesbecome brighter since the function emphasizes bright objects.

In order to collect data at a much faster rate and increase the amountof solution which is sampled, it is advantageous to scan the solution,either by moving the incident illuminating beam, or moving the solution.One can periodically sample at hundreds or thousands of locations withinthe solution using in each case a sample volume of the same size,typically δV≈10⁻⁶ cm³. The correlation functions for the sample volumesare added together since the information from each sample volume isequivalent. The scanning therefore increases the rate at which oneobtains the correlation function.

The baseline of the correlation function, which with one sample volumewas obtained by calculating the correlation function at very long sampletimes (region 200 of FIG. 10), can also be computed by measuring thecorrelation function between separate volume elements, i.e.

    Baseline=<I(t)-I'(t')>

where I(t) is the intensity, at time t, from one sample volume andI'(t') is the intensity at time t' from another sample volume. It is notnecessary that t' be different than t, but in a system which isscanning, only one intensity is measured at any given time. Since thefluctuations in the number of particles in different sample volumes arestatistically independent, this correlation function will produce justthe square of the average intensity, which is the baseline of theautocorrelation function.

The correlation function at zero sampling time, minus the baseline, forsuch a scanning system would be the square of the intensity from eachsampling volume, averaged over sampling volumes, minus the square of theaverage intensity, i.e.

    C(0)-B=<I(t').sup.2 >.sub.t' -<I(t')>.sup.2.sub.t'

This is the mean square fluctuation of the intensity from samplingvolume to sampling volume, This fluctuation increases as more of thefluorescence becomes bound to the carrier particles, independent of thediffusion rates of the free and bound fluorescence.

Measuring the fluctuation in intensity from sample volume to samplevolume as a means of monitoring changes in the fluorescence from thefree to bound state simplifies the scanning system in that one does notneed to return to each sample volume at a later time since only thecorrelation function at zero sampling time is measured. This wouldsimplify the apparatus whether one were moving the incident illuminatingbeam or moving the solution since one would not have to "keep track" ofwhere the sample volumes are and would allow one to scan at higher ratesand reduce the time required for the measurement.

As mentioned above, an experimental apparatus which was designed to makea correlation-based immunoassay measurement is shown diagrammatically inFIG. 4. The design to be described is a particular representation and isshown by way of example only. A pinhole 60, was used to define a narrowbeam of (exciting) light for the purpose of illuminating a portion ofthe sample solution contained in sample cell 62. It was useful to employa pinhole of diameter 100 microns (0.1 mm); other values may be chosento be more appropriate, depending on the details of the sample solution.(discussed below). Initial experiments to assay the presence of theimmunoglobulin, IgG, utilizes the antibody to IgG which was labeled withthe fluorescent tag fluorescein isothiocyanate (FITC). It was convenientto employ an argon-ion laser having a collimated output beam ofwavelength equal to 488 nanometers (blue). However, coherence of theexciting light source is not required for the present invention; anysuitably filtered incandescent or other light source of appropriateintensity will suffice. The range of exciting wavelengths passed by thefilter should ideally be substantially shorter than the useful emissionwavelength range (yellow green for FITC) of the fluorescently taggedmolecules.

Any sources of fluorescence in the sample solution which lie within thenarrow beam volume defined by pinhole 60 in FIG. 4 will emit fluorescentlight. These sources include fluorescently tagged molecules plusnaturally occurring fluorescing species (e.g. contamination). The lens64 projects this fluorescing source image onto slit 68 at the face ofthe photomultiplier 70. The filter 66 is used to block any 488 nanometer(blue) exciting light which may be scattered from the sample solution.The resulting light intensity which reaches the photomultiplier tube 70will be the yellow green fluorescein emission wavelengths, together withany other long wavelengths emitted by fluorescing background impurities.It was convenient to fix the width of slit 68 at approximately 100microns. Thus, the effective sample volume which was both illuminatedand detected was on the order of 10⁻⁶ cubic centimeters, as defined bythe optics employed.

The sample cell 62 is a cylindrical tube which is uniformly translatedin an up/down vertical motion using a loud speaker or other suitableelectromechanical transducer. Two sizes of sample tubes were used in theexperiments; 6 millimeter outer diameter disposable culture tubes andtwo millimeter outer diameter 100 microliter micropipets. These tubeswere centered within a standard one centimeter fluorimeter cuvettefilled with water for the purpose of optical index matching with theincident light beam to minimize stray reflections. Sample solutionvolumes as small as 20 microliters are easily achieved in the smalltubes.

The sample tube was translated up to 0.8 centimeter according to atriangular wave form, with the period adjustable from 0.2 to 20 seconds.The triangular driving wave form was made up of a rising and fallingstaircase of discrete steps, adjustable in number from 64 to 1024. Eachstep corresponds to a discreet sample volume. A microcomputer, such asthe Motorola 6800, was employed to synchronize the sample position withthe calculation of the correlation function C(t). The computer muststore the measurements for one complete cycle of the scan so that it cancalculate the product between the present intensity from some samplevolume and the intensity obtained from that same sample volume on theprevious scan, i.e.

    I(t)·I(t-τ)

To obtain the baseline of the correlation function, the computer alsocalculates for each sample

    I(t)·I'(t-t')

where t'≠τ.

These two products are then averaged over all samples to obtain theautocorrelation function:

    C(τ)=<I(t)·I(t-τ)>.sub.+

and its baseline value

    C'(t')=<I(t)·I'(t-t')>.sub.+

The scanning time was chosen to be larger than the diffusion time of thefree tagged antibodies and shorter than the diffusion time of thecarrier particles.

The ability of the correlation assay to determine the amount of taggedIgG bound to the carrier particles in the presence of varying amounts offree fluorescently tagged antiserum or antibodies was the subject ofadditional tests, the results of which are shown in FIG. 7. Thediffusional lifetime of the four micron diameter acrylamide beads wasestimated to be roughly 6,000 seconds. The corresponding lifetime forthe fluorescently tagged free molecules in solution deduced from dynamiclight scattering was determined to be approximately 25 seconds.Accordingly, in order to use diffusion times to discriminate against thefree tagged molecules, one should employ a scanning period τ in theorder of 100 seconds.

However, we found that the fluctuation lifetime of all species, largeand small, were limited to about 10 seconds by, it is assumed, velocitycurrents in the sample, which may be at least partially a result of theacceleration of the up/down motion of the sample cell in theconfiguration shown in FIG. 4 which was being employed. Accordingly, forthe tests involving the system of FIGS. 6a, 6b and 6c, it was necessaryto use a sufficiently low repetition rate to insure that at least thelarger particles would contribute significantly to the correlatedfunction, and a period of 1.2 seconds was selected. The plot 116 in FIG.7 shows the correlation results, expressed in relative units, as afunction of the amount of the free fluorescently tagged anti-serum orantibody, normalized to the fluorescent intensity of the boundfluorescent molecules. In the tests of FIG. 7, the IgG concentration andthe carrier particle density were maintained fixed. From curve 116, wemay note that when the background free fluorescence is 10 times thecarrier particle fluorescence (see points 117 and 119 on curve 116),there is virtually no change in the correlation result for boundfluorescence. Thus, experimental confirmation has been obtained that thecorrelation assay does achieve a level of selectivity even when bothfluorescing species have long enough lifetimes to contribute to thecorrelation. It is again noted that the rapid movement of the sampledecreased the fluctuation lifetime of both types of particles and thatthe sample time of 1.2 seconds was not the figure which would have beenpreferred in the absence of the fluid current effect.

These results point out the property of the correlation technique thatthe correlation function goes as I² N and is therefore more sensitive toa small number of bright objects (carrier particles) than a large numberof dim objects (individual free fluorescent molecules) even though theaverage intensity, IN, from both species is equal. So even withoutdepending on a difference in diffusion times between the bound and freefluorescence, the technique enhances the detection of fluorescence whichis bound to the carrier particles.

The plots 118 and 120 represent the results of further tests in whichthe fluctuation lifetime of a smaller (free) fluorescing species isdiffusionally limited. In the tests which resulted in curves 118 and120, commercially available fluorescent spheres having a diameter of 4.3micrometers were employed for the large species and the dye rhodamine 6Gwas employed for the fast-diffusing species. The latter has a molecularweight of 530 with an estimated hydrodynamic radius of approximately 6.7Angstroms. In addition, in order to reduce flow currents due to samplemotion and thermal gradients, the cell sample size was drasticallyreduced. More specifically, a portion of a standard 100 microlitermicropipet was employed, which had a 2 millimeter outside diameter,resulting in an active solution volume of less than 20 microliters. Thiswas centered in a fluorimeter cuvette filled with water for indexmatching, as well as temperature control. The use of the small diametertube coincidentally reduced the volume dV by a factor of about 3 due tothe focusing lens effect of the small glass tube. The resultingdiffusional relaxation time out of this reduced volume for thepolystyrene spheres and the rhodamine 6G were estimated to be about3,000 seconds and 1.0 seconds, respectively. Thus, we expected that ascanning period in the order of 10 seconds should allow us todiscriminate effectively against the background fluctuations of therhodamine 6G whereas at a scanning period τ of one second, only marginaldiscrimination was expected.

These anticipated results are confirmed by plot 118 using a timeinterval τ of 1.2 seconds and plot 120 using a time interval of 10.2seconds. It is particularly to be noted that in connection with plot120, very little deviation in the output signal occurred even at ratiosof free-to-bound fluorescent molecules well above 100. This analysisclearly demonstrates the high accuracy of this immunoassay techniquewhen fast diffusing sources of fluorescence are employed, and whereconditions are controlled so that it is practical to select a scanningperiod which is between the diffusion relaxation time for the larger andsmaller particles.

FIG. 8 of the drawings illustrates the utilization of the so-called"shadow" effect which may be used to differentiate between freefluorescent molecules as compared with bound fluorescent molecules. InFIG. 8 a large carrier particle 124 is shown being illuminated by a bluebeam of collimated light 126, with fluorescent radiation being picked upin both the forward direction along path 128 through filter 130, and inthe rear or back direction along path 132 through filter 134. With allof the fluorescent molecules being bound to carrier particles such as124, and with these particles being relatively large and opaque, such asfine carbon particles, or metal coated particles, very littlefluorescent illumination will be observed along path 128; but a highsignal level of fluorescent radiation will be observed along path 132 asa result of the full exposure of the left-hand side of the largeparticle 124 to the input radiation 126. Fluorescent light from freefluorescent molecules will not be shadowed in the forward direction.

The fluorescent intensity measured along the path 128 is a measure ofthe number of free fluorescent molecules whereas the difference betweenthe fluorescent intensity measured along path 132 and the fluorescentintensity measured along path 128 is a measure of the number offluorescent molecules bound to the opaque carrier particle. If thecarrier particles were semi-opaque, one may apply correction factors tothe intensity measurements to obtain a measure of either the number ofbound fluorescent molecules or the number of free fluorescent molecules.Mirrors and a beam chopper may also be employed to alternately measurethe intensity along paths 128 and 132 with a single photodetector.

In addition, of course, the signals derived as indicated in FIG. 8 maybe employed with the autocorrelation technique to provide theenhancement of detection accomplished by both of these techniques toprovide an extremely sensitive immunoassay technique. Further, thesystem of FIG. 3 with its forward and back photomultiplier detectors andits data processor 46 including an auto-correlation capability, may beemployed to implement this combined shadowing and autocorrelationsystem.

Background literature which is useful in the understanding andconsideration of the present invention includes an article onradioimmunoassays, entitled, "A Physicist in Biomedical Investigation",by Rosalyn S. Yalow, Physics Today, October 1979, pages 25 through 29;and an article entitled "Determination of Molecular Weights byFluctuation Spectroscopy: Application to DNA", by M. Weissman et al.,Proceedings of the National Academy of Science, U.S.A., Volume 73, No.8, pages 2776 to 2780, August 1976; and a brochure entitled,"Immuno-Fluor", dated May 1978 and published by BIO-RAD Laboratories,2200 Wright Avenue, Richmond, Calif. 94804. Incidentally, theImmuno-Fluor pamphlet describes the separation type immunoassay usingfluorescent material which has been mentioned hereinabove in the presentspecification. Concerning the Weissman et al. article, it discloses theuse of fluctuations in intensity from sampling point to sampling pointto determine the number density of large fluorescent particles insolution, but there is no examination of a process or the results of aprocess involving the shift of fluorescently tagged molecules fromsolution to large carrier particles of the type described hereinabove inthe present specification.

In conclusion, it is to be understood that the foregoing description andthe drawings are illustrative of specific techniques of the invention.Further, the principles of the invention are clearly applicable to otherimmunoassay systems and to other methods of analysis wherein thereaction involves the transfer of fluorescent material between boundstates on carrier particles of a large size and a "free" state where thefluorescent particles are not bound to the large carrier particles.Other electronic, optical, and mechanical techniques and systems may beemployed to implement the principles of the invention. Accordingly, thisinvention is not limited to that precisely as shown and describedhereinabove.

What is claimed is:
 1. A homogeneous fluorescent immunoassay methodcomprising the steps of:providing relatively large carrier particles insolution with antibodies residing at a plurality of sites on each ofsaid carrier particles; providing at least first and second activecomponents; with the first being an unknown antigen to be tested, thesecond being tagged with a fluorescent substance; exposing said carrierparticles to the first and second components, to cause a reactionbetween said first and second components and the antibodies on saidparticles; whereby the number of fluorescently tagged components bondedto said carrier particles changes; illuminating the solution containingboth the free and bound fluorescently tagged substances with opticalradiation at a first wavelength to cause fluorescent output radiation ata second longer wavelength; optically sensing output signals at saidsecond wavelength resulting from said fluorescent output radiation; andelectronically processing the optically sensed signals to determine theamount of the fluorescently tagged component which is free and/or thatwhich is bound to the carrier particles without physically separatingthe free and bound fluorescent material, said electronic processingincluding autocorrelation processing to discriminate between fluorescentradiation arising from the bound fluorescent material and from the freefluorescent material.
 2. A fluorescent immunoassay method as defined inclaim 1 wherein the autocorrelation function at zero sampling time isused.
 3. A fluorescent immunoassay method as defined in claim 1including the step of selecting a very small volume for sampling toinclude a relatively small number of the larger carrier particles, sothat the relative fluctuation in the number of such larger particleswill be large.
 4. A fluorescent immunoassay method as defined in claim 1wherein the electronic processing includes measuring the fluctuations inthe fluorescent radiation to emphasize the fluorescence from thebrighter carrier particles.
 5. A fluorescent immunoassay method asdefined in claim 1 in which the second component is an antigen of thesame kind as the first component and the carrier particle is exposed toboth the first and second components which then compete for bindingsites on the carrier particle.
 6. A fluorescent immunoassay method asdefined in claim 1 in which the second component is an antibody and thecarrier particle is initially exposed to the first component and then tothe second component.
 7. A fluorescent immunoassay method as defined inclaim 1 wherein said carrier particles are biological entities havingnatural reactive sites.
 8. A fluorescent immunoassay method as definedin claim 1 including the steps of using opaque carrier particles, andsensing fluorescent radiation from the "forward" direction relative tosaid first wavelength radiation, whereby the carrier particles shield or"shadow" the radiation from the bound fluorescent molecules.
 9. Afluorescent immunoassay method as defined in claim 8 wherein thefluorescent radiation is also sensed in the "back" direction, to obtainfull fluorescent radiation from the bound fluorescent molecules, andfurther including the steps of comparing the signals sensed in the"forward" and in the back directions to determine the relative levels ofbound and free fluorescent molecules.
 10. A fluorescent immunoassaymethod as defined in claim 1 including the steps of selecting a seriesof very small volumes to include a relatively small number of said largecarrier particles in each volume, and sampling the fluorescence fromeach said volume.
 11. A fluorescent immunoassay method as defined inclaim 10 wherein each of said volumes is only sampled once prior todetermining the amount of the free and the bound fluorescently taggedcomponent.
 12. A fluorescent immunoassay method as defined in claim 10wherein each of said volumes is sampled a plurality of times prior todetermining the amount of the free and the bound fluorescently taggedcomponent.
 13. A homogeneous fluorescent immunoassay method comprisingthe steps of:providing carrier particles of a relatively large size insolution and with a large number of reactive sites on the surface ofeach particle; providing at least first and second active components;with one being of known properties and composition and the other beingan unknown to be tested; with one being tagged with a fluorescentsubstance and the other being untagged; and said first and secondcomponents including antigens and antibodies; exposing said carrierparticles to said first and second component, to cause a reactionbetween said first and second components and the reactive sites on thecarrier particles; whereby the number of fluorescently tagged antibodiesor antigens bonded to said carrier particles changes; illuminating thesolution containing both the free and bound fluorescently taggedsubstances with optical radiation at a first frequency to causefluorescent output radiation at a second different frequency; opticallysensing output signals at said second frequency, resulting from saidfluorescent output; electronically processing the optically sensedsignals to determine the amount of the fluorescently tagged componentwhich is free and which is bound to the carrier particles withoutphysically separating the free and bound fluorescent material; andselecting a very small volume for sampling to include a relatively smallnumber of the larger carrier particles so that the relative fluctuationin the number of such large particles in the volume causes a bigfluctuation in output fluorescence.
 14. A fluorescent immunoassay methodas defined in claim 13 wherein said electronic processing includesautocorrelation processing to disciminate between fluorescent radiationarising on the larger particles and the fluorescent radiation fromsmaller "free" fluorescent molecules.
 15. A fluorescent immunoassaymethod as defined in claim 13 including the step of filtering thefluorescent radiation at said second frequency prior to optical sensingto eliminate scattered or direct radiation at said first frequency. 16.A fluorescent immunoassay method as defined in claim 13 including thestep of selecting a series of very small volumes for sampling to includea relatively small number of said larger carrier particles in eachsample, so that variations in the number of said larger particles insaid volumes will cause large fluctuations in the sensed outputfluorescence.
 17. A fluorescent immunoassay method as defined in claim13 wherein the electronic processing includes measuring the fluctuationsin the fluorescent radiation to emphasize the fluorescence from thebrighter carrier particles.
 18. A fluorescent immunoassay method asdefined in claim 17 where the autocorrelation function at zero samplingtime is used.
 19. A fluorescent immunoassay method as defined in claim13 including the steps of using opaque carrier particles, and sensingfluorescent radiation from the "forward" direction relative to saidfirst frequency radiation, whereby the carrier particles shield orshadow the radiation from the bound fluorescent molecules.
 20. Afluorescent immunoassay method as defined in claim 19 wherein thefluorescent radiation is also sensed in the "back" direction, to obtainfull fluorescent radiation from the bound fluorescent molecules, andfurther including the steps of comparing the signals sensed in the"forward" and in the back directions to determine the relative levels ofbound and free fluorescent molecules.